Blocking of amino acid transporter OsAAP7 promoted tillering and yield by determining basic and neutral amino acids accumulation in rice

Background Amino acids are not only the main form of N in rice, but also are vital for its growth and development. These processes are facilitated by amino acid transporters within the plant. Despite their significance, only a few AAP amino acid transporters have been reported. Results In this study, we observed that there were differences in the expression of amino acid transporter OsAAP7 among 521 wild cultivated rice varieties, and it directly negatively correlated with tillering and grain yield per plant. We revealed that OsAAP7 protein was localized to the endoplasmic reticulum and had absorption and transport affinity for amino acids such as phenylalanine (Phe), lysine (Lys), leucine (Leu), and arginine (Arg) using subcellular localization, yeast substrate testing, fluorescent amino acid uptake, and amino acid content determination. Further hydroponic studies showed that exogenous application of amino acids Phe, Lys and Arg inhibited the growth of axillary buds in the overexpression lines, and promoted the elongation of axillary buds in the mutant lines. Finally, RNA-seq analysis showed that the expression patterns of genes related to nitrogen, auxin and cytokinin pathways were changed in axillary buds of OsAAP7 transgenic plants. Conclusions This study revealed the gene function of OsAAP7, and found that blocking of amino acid transporter OsAAP7 with CRISPR/Cas9 technology promoted tillering and yield by determining basic and neutral amino acids accumulation in rice. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05159-5.


Introduction
Oryza sativa (Rice) is the primary grain crop globally, particularly in Asia [1].The swiftly increasing population will be met with a rise in the demand for rice production, making it crucial to enhance rice grain yield to address this future challenge.The grain yield potential of rice is influenced by various components, with tillering being a key factor [2].In rice plants, tillers originate from basal internodes, and each tiller is crowned with a panicle [3].Furthermore, tillering influences the panicle number per plant, ultimately regulating grain yield [4].
Nitrogen (N) is a vital nutrient for crop growth and can regulate the process of rice tillering [5,6].Plants can uptake both inorganic nitrogen (nitrate and ammonium) and organic nitrogen (amino acids) from the soil.Subsequently, a significant portion of nitrate and a fraction of ammonium undergo assimilation into amino acids.Additionally, amino acids serve as the primary N form for transportation and allocation in rice [7].Therefore, it is crucial to investigate whether and how amino acids affect rice growth, particularly for tillering.
Amino acid permease (AAP) is a type of amino acid transporter capable of absorbing and transporting amino acids [9].To date, many AAPs have been functionally studied in higher plants.In Arabidopsis, AtAAP1 primarily uptakes glutamate (Glu), histidine (His), and phenylalanine (Phe); the mutant aap1 can tolerate high levels of amino acids to avoid the suppression of plant growth [10].Moreover, it has been demonstrated that AtAAP1 is essential for the synthesis of storage protein and formation of seed yield [11]. AtAAP2 is involved in the xylemphloem transfer of amino acids.Interestingly, the aap2 mutant exhibits improved characteristics in terms of branch number, silique number, seed yield, and nitrogen use efficiency (NUE) compared to those of the wild-type [12,13].AtAAP3 is mainly expressed in the phloem [14].The AtAAP6 mutant has a larger rosette width, number of cauline leaves, and seed size compare with the wildtype [15].Additionally, AtAAP8 plays an important role in seed development and influences seed number [16].
In rice, 19 AAPs have been identified [17].Among these genes, OsAAP1 facilitates the transport of neutral amino acids and positively regulates plant growth and yield [18]. Besides, OsAAP3 negatively controls bud outgrowth, tiller number and grain yield, and reducing OsAAP3 expression can enhance NUE in rice [19].Overexpression of OsAAP4 increases tiller number and grain yield by transporting valine (Val), proline (Pro), threonine (Thr) and leucine (Leu) [20].OsAAP5 negatively affects bud outgrowth through cytokinin content mediation [21].OsAAP6 regulates the uptake and distribution of amino acids in rice [22].Amino acid transporter OsAAP14 determines bud outgrowth under melatonin treatment, thereby influencing rice tillering and grain yield [23]. Overexpressing of OsAAP15 in rice enhances both primary and secondary branches in the panicle, attributed to its impact on the concentrations of amino acids Tyr and Val in the rice panicle [24].In maize, ZmAAP6 positively modulates grain protein [25].
Gene editing technology encompasses the methodology aimed at modifying specific sites within a target gene sequence [26].CRISPR/Cas9 technology represents one of the most recent advancements in gene editing with widespread applications in elucidating gene functions across various organisms compared to ZFN and TALEN technologies [27].The CRISPR/Cas9 gene editing process typically involves two primary steps.Firstly, a specific guide RNA (sgRNA) is employed to target the gene sequence of interest.This sgRNA is synthesized in vitro, utilizing a design tailored to the specific DNA sequence being targeted.Secondly, once the sgRNA has successfully bound to its complementary sequence within the target DNA, the Cas9 protein, which acts as a molecular scissors, facilitates the editing process.Cas9, guided by the sgRNA, precisely cuts the DNA at the intended location, thereby initiating the editing process.In essence, the CRISPR/Cas9 system utilizes the sgRNA to locate the target sequence through base pairing, enabling Cas9 to perform the precise DNA editing [28].This approach has revolutionized the field of genetic engineering due to its simplicity, efficiency, and versatility.
For RNA-seq, it has evolved alongside second-generation high-throughput sequencing (NGS) [29].The transcriptome comprises all transcripts produced under specific temporal and spatial conditions, encompassing mRNA and non-coding RNA.It serves as the bridge between genetic information and the biological functions of the proteome [30].RNA-seq technology not only enables the study of gene function and structural characteristics at the transcriptional level but also unveils the molecular mechanisms involved in biological processes under specific conditions [31,32].Presently, the primary experimental process of high-throughput RNA-Seq sequencing technology includes total RNA extraction from samples, obtaining high-quality mRNA, and synthesizing cDNA using mRNA as a template, followed by a series of purification, elution, repair, recovery, and PCR reaction to construct the entire sample library, quality detection through instruments, and qRT-PCR.Finally, the qualified library undergoes sequencing, after which the sequencing data are analyzed to complete the entire technical process.
In this study, our aim was investigation of the function of OsAAP7 AAPs using CRISPR/Cas9 and RNA-seq technology to elucidate the physiological mechanism of OsAAP7 on amino acid absorption and transport in rice.Our findings have significant implication for rice highyield breeding program with gene editing technology.

Material and method
Sequence variation of OsAAP7 promoter SNP variants in the promoter of OsAAP7 (LOC_ Os05g34980) were extracted from 521 wild rice varieties worldwide, as provided by RiceVarMap 2.0 (website: http://ricevarmap.ncpgr.cn/)[33].The 521 wild rice varieties were cultivated in experimental paddy fields in Huaxi District, Guizhou Province, spanning from May to October.The applied fertilizer rate was 270 kg•ha − 1 , with a nitrogen, phosphorus and potassium ratio of 19%, 7% and 14%, respectively.Three haplotypes (Hap1-Hap3) of OsAAP7 were identified, and the expression of OsAAP7 corresponding to Hap1-Hap2 was determined using realtime fluorescence quantitative PCR (qRT-PCR).Primers were designed with Primer Premier 5 and were provided in Supplemental Table S1.Subsequently, associations between the two haplotypes and agronomic traits were examined.

Generation of OsAAP7 plant vector
To generate an overexpression vector of OsAAP7, the cDNA (1491 bp) of OsAAP7 was fused to pCAMBIA1306 vector under 35 S promoter with BamH I and Kpn I, and introduced into wild-type ZH11.Due to the SNP mutation sites on exon 4 of OsAAP7, we employed double targets to knockout the fourth exon of the OsAAP7 sequence in ZH11 to improve knockout efficiency with CRISPR/ Cas9 technology.For the construction of the OsAAP7 promoter-GUS vector, a promoter (2113 bp) fragment upstream of the OsAAP7 coding region was amplified and inserted into pCAMBIA1391Z vector upstream of β-glucuronidase (GUS) gene, utilizing BamH I and EcoR I enzymes.All vectors were subsequently transformed into Agrobacterium, and transgenic lines were generated by infecting calli from japonica rice ZH11 with Agrobacterium [34].The T2-generation was selected for subsequent experiments.Primer design was carried out using Primer Premier 5 and SnapGene, with details in Supplemental Table S1.

Rice growth conditions and agronomic traits analysis
ZH11, OsAAP7 overexpression (OE) and CRISPR (C) lines were planted in the Huaxi and Lingshui experimental fields of Guizhou University with a fertilizer application of (N/P/K = 19%/7%/14%) 270 kg•ha − 1 .During the rice maturing stage, 30 plants of each line were counted for tiller number and grain yield.T2-overexpressing transgenic lines of OsAAP7 were detected by qRT-PCR, and CRISPR lines of OsAAP7 were identified using sequencing.All primers were designed using Primer Premier 5 with details in Supplemental Table S1.

Subcellular localization
The cDNA sequence of OsAAP7 was amplified and fused with the GFP (Green Fluorescent Protein) of HBT plasmid.Subsequently, the recombinant vector was transiently transformed into rice protoplasts.AtWAK2, which is located on the endoplasmic reticulum membrane, was used with mCherry as a membrane localization marker.Fluorescence was detected with confocal microscope (Nikon, Japan).The information of all primers was designed by Primer Premier 5 with details in Supplemental Table S1.

GUS staining and paraffin-slicing analysis
To obtain OsAAP7 expression pattern, GUS staining of different tissues of OsAAP7 promoter-GUS transgenic lines was executed with GUS Blue Kit (Huayueyang, China).GUS staining was performed according to a previously described histochemical staining method [5].Then, the photos were taken by tissues with stereomicroscope (Olympus, Japan).For paraffin-slicing analysis, the solution of 50% ethanol: 10% formaldehyde: 5% acetic acid was added in the tissues, then water in gradient 50-100% ethanol was reduced, and finally it was buried in paraffin.These samples were sectioned with slicing instrument (Leica, Germany) and were examined with microscope (Zeiss, Germany).

Yeast complementation assays of OsAAP7
The empty vector and pDR196-OsAAP7 construct were transformed into 22Δ10α of yeast mutant (Saccharomyces cerevisiae).The wild-type strain 23344c served as a control to monitor yeast growth.Yeast transformation was carried out using the Yeast Transformation Kit (Coolaber, China).Transformants were then incubated at 30 °C for 2-4 d after plating onto screening medium plates (SD-Ura solid medium).Colonies were subsequently grown in YPDA liquid medium until the optical density (OD) at 600 nm (Ab600) reached approximately 0.6-1.0.The yeast culture was then centrifuged at 5000 rpm for 2 min in a 1.5 mL centrifuge tube, then the supernatant was discarded, and the pellet was washed with ddH 2 O 3-4 times.The yeast culture was diluted with ddH 2 O to a specific absorbance value.7 µL of each yeast solution at different dilutions was placed on uracil-free YNB solid medium (lacking amino acids, NH 4 + and uracil; Coolaber, China), 3 mM (NH 4 ) 2 SO 4 or 3 mM of various amino acids was added alone as the sole N source.The yeasts were then incubated with sterile water at dilutions of 1, 10, 100 and 1000 to achieve OD600 values of 0.1, 0.01, 0.001, and 0.0001, respectively.The plates were inverted and placed in a thermostat at 30 °C.After being inverted and incubated at 30 °C for 2-3 d, they were observed with camera.

Total N and amino acid determination
Total N concentration was determined with a total nitrogen analyzer (SKD-100, Peiou, China).N utilization efficiency (NUtE, %) was calculated with equation: [Grain yield (g) / (Grain N concentration (g) + Straw N concentration (g))] × 100.The concentrations of individual free amino acids were measured as follows: 1 g of sample was incubated in 10 mL of 80% ethanol at 80 °C for 20 min, then the supernatant was transferred with repeated steps.The solution was dried to remove ethanol and water at 80 °C, and 1 mL of 0.5 mM NaOH was added.After centrifugation at 14,000 rpm for 20 min, the supernatant was filtered using a 2 μm filter membrane.Finally, the filtrate containing amino acids was analyzed using HPLC Agilent-1260 (Agilent, America).

Fluorescent amino acid uptake experiment
To delve deeper into amino acid transport, ZH11, OsAAP7 OE and C lines were subjected to treatment with 1 mM Fluorescent amino acid Arg, Phe or Lys for 2 h, 6 or 10 h.Subsequently, the rice seedlings were examined using a Chemiluminescence Apparatus (Qinxiang, China), and the fluorescent intensity was quantified utilizing Bandscan [23,24].

Hydroponic culture and seedling growth observation
For hydroponic culture, rice seedlings of ZH11, OsAAP7 OE and C lines were initially grown in basic rice nutrient solution composing of 1 mM NH 4 NO 3 , 0.32 mM NaH 2 PO 4 , 0.51 mM K 2 SO 4 , 1.0 mM CaCl 2 , 1.65 mM MgSO 4 , 8.9 µM MnSO 4 , 0.5 µM Na 2 MoO 4 , 18.4 µM H 3 BO 3 , 0.14 µM ZnSO 4 , 0.16 µM CuSO 4 , and 40.0 µM FeSO 4 for 1 week.Then the seedlings were transferred to a solution supplemented with 1.0 mM NH 4 NO 3 and one of the amino acids Arg, Phe or Lys.The lengths of both the first and second axillary buds were assessed with stereomicroscope (Olympus, Japan).The rice seedlings were grown in boxes (525 mm×360 mm×230 mm) with the solution in a phytotron under condition of 30 °C for 14 h and 25 °C for 10 h.The culture solution was replaced every 3 d.

RNA-seq analysis
Axillary buds of sixty seedlings were collected from ZH11, OsAAP7 OE1 and C1 lines each with a replicate weight of 0.5 g.RNA-seq analysis was conducted by Personalbio (Personalbio, China).The resulting data are accessible at the National Center for Biotechnology Information (NCBI).Clean data were aligned to the rice genome sequence (Oryza sativa.IRGSP-1.0)[35].Reads were counted and summarized using feature counts [36].Differentially expressed genes (DEGs) were identified utilizing the DESeq package, with a significance thresholds of p value < 0.05, and a fold-change > 1.4 set as criteria for significant differential expression.

Statistical analysis
The statistical charts were created using GraphPad Prism 8, while the heatmaps were generated using TBtools.To assess statistical differences, a t-test was employed with SPSS (IBM, Inc.).Significance levels were denoted as follows: *, **, and *** represented significant differences at P < 0.05, P < 0.01, and P < 0.001, respectively.Additionally, Tukey-Kramer's multiple range test was conducted using SPSS software, with significant difference indicated at P < 0.05.The data are presented as mean ± standard deviation values.

Promoter of amino acid transporter OsAAP7 was divergent between indica and japonica rice
Promoter of amino acid transporter OsAAP7 (2600 bp upstream of the codon ATG) was investigated in 521 wild rice varieties worldwide utilizing haplotype (Hap) analysis with the Rice Variation Map v2.0 [37].The findings revealed three major Haps among these cultivars (Fig. 1A).Notably, Hap1 predominated in indica, whereas Hap2 was prevalent in japonica (Fig. 1A).Analyzing the relative expression of OsAAP7 among the three haplotypes unveiled a distinct expression pattern in the japonica haplotype (Hap2) compared to the indica haplotype (Hap1; Fig. 1B).Furthermore, it was observed that indica haplotype exhibited more tillers than japonica (Fig. 1C).The yield of Hap1 surpassed that of Hap2 (Fig. 1D), and the total weight of Hap1 was significantly higher than that of Hap2 (Fig. 1E).Additionally, a comparison of OsAAP7 expression in randomly selected 10 indica and 10 japonica wild cultivars among with Hap1 and Hap2, demonstrated lower expression in indica with Hap1 than in japonica with Hap2 (Fig. 1F).Conversely, indica with Hap1 displayed more tillers than japonica with Hap2 (Fig. 1G).These results underscored differences in the promoter sequences of OsAAP7 between indica and japonica.Consequently, indica varieties with Hap1 exhibited increase of tillering but decrease of OsAAP7 expression compared to japonica varieties with Hap2, implying a potential correlation between elevated OsAAP7 expression and reduced rice tillering.

Expression pattern of OsAAP7 at different stages in rice tissues, and its subcellular localization
To identify the expression pattern of OsAAP7, pOsAAP7-GUS transgenic lines were established, and various tissues both during vegetative and reproductive stages were subjected to GUS staining.The GUS-staining experiments revealed that OsAAP7 expression was elevated in lateral roots, leaf sheaths and leaf blades (Fig. 2B, D, E) during vegetative stage but was diminished in root tips (Fig. 2A) and stems (Fig. 2C).Conversely, during the reproductive stage, OsAAP7 expression heightened in stems (Fig. 2G) and leaf sheaths (Fig. 2H) but declined in roots (Fig. 2F), leaf blades (Fig. 2I), and young panicles (Fig. 2J).qRT-PCR analysis further corroborated the expression profiles of OsAAP7 during the vegetative stage (Fig. 2K) and reproductive stage (Fig. 2L).The vascular tissues and parenchyma cells of leaf blades (Fig. 2N) and leaf sheaths (Fig. 2O) exhibited higher GUS activity during the reproductive stage, whereas roots (Fig. 2M) and young panicles (Fig. 2P) displayed lower GUS activity.
Besides, it was revealed that the expression of OsAAP7 in lateral roots was elevated after a 2 h treatment with basic amino acids by GUS staining.Furthermore, a higher expression of OsAAP7 in lateral roots was observed by 8 h treatment with acidic or neutral amino acids (Supplementary Fig. S1A).Subsequent qRT-PCR analysis further confirmed the expression profile of OsAAP7 under treatment with basic, acidic and neutral amino acids (Supplementary Fig. S1B).
Furthermore, the study demonstrated that the GFP control exhibited fluorescent proteins distributing in both the plasma membrane and nucleus within the rice protoplasts (Supplementary Fig. S2A).In contrast, the fluorescence pattern of OsAAP7-GFP was notably concentrated in the endoplasmic reticulum (ER) membrane, consistenting with the localization of a co-marker which also distributed in the ER membrane.This observation suggests that the OsAAP7 protein is predominantly localized within the endoplasmic reticulum membrane (Supplementary Fig. S2B).

Substrate transport of OsAAP7 protein with yeast complementation assay and fluorescent amino acid uptake experiment
To ascertain OsAAP7 protein's role in amino acid transport, we examined substrate transport using the mutant yeast strain 22Δ10α.Medium containing 3 mM amino acids Phe, Lys or Leu was crucial for the growth of 22Δ10α-transformed OsAAP7 protein (controlled by pDR196), respectively (Fig. 3A).To assess OsAAP7 protein is transport properties, yeast cells expressing the transporter were cultured in liquid culture with 3 mM Phe, Lys or Leu as the sole N source (Fig. 3B).Results indicated that the growth rate of cells expressing OsAAP7 in pDR196 was faster than those of empty pDR196 vector for these amino acids, monitoring with absorbance of 600 nm light (Fig. 3B).Previous substrate testing also revealed OsAAP7 protein had ability to transport amino acid Arg [38].These findings suggest that OsAAP7 protein predominantly transports amino acids Phe, Lys, Leu and Arg.

OsAAP7 negatively regulated grain yield in rice
To investigate the impact of OsAAP7 expression changes on rice growth, we generated OsAAP7 overexpression (OE) lines under the 35 S promoter and OsAAP7 mutant (C) lines using CRISPR/Cas9 technology.We obtained 24 T0 generation seedlings of OE and 24 T0 generation seedlings of C lines with via the Agrobacterium-mediated method.Sequencing of these C lines mutants revealed a total of 11 plants with 860 bp deletion, 2 bp deletion and 1 bp insertion mutation (Supplementary Fig. S3A, S4).It resulted in transformation of 19 mutant lines with verification of the Cas9 protein-encoding gene (Supplementary Fig. S3B).Subsequently, seven mutant lines from the T0 generation were selected and propagated to the T1 generation, and three mutant lines were confirmed through sequencing (Supplementary Fig. S3C, S5).The absence of the Cas9 protein was confirmed in the T2 generation (Supplementary Fig. S3D).
The same procedure was followed to verify the hygromycin gene in 24 T0 generation OE lines, leading to the successful transformation of 12 lines (Supplementary Fig. S3E).The expression of the OsAAP7 gene in these 24 lines is shown in Supplementary Fig. S6.Among these, 6 lines with the higher expression were selected and propagated to the T1 generation to confirm OsAAP7 expression and the presence of the hygromycin gene (Supplementary Fig. S3F, S7).The expression of OsAAP7 was verified in OE lines, and the sequence of OsAAP7 was confirmed in C lines, respectively in T2 generation (Fig. 4C, D).Finally, these three different C lines (C1-C3 derived from T0-9, T0-12, and T0-16) and three OE lines (OE1-OE3 derived from T0-11, T0-14, and T0-22) in T2 generation were chosen for subsequent experiments (Supplementary Fig. S3G, S8).
To assess the impact of altered OsAAP7 expression on traits, we determined tiller number and grain yield per plant of OE and C lines in paddy field.We observed a reduction in tiller number per plant in OE lines compared to the wild-type, whereas an increase was noted in C lines (Fig. 4A, E).Additionally, in comparison with ZH11, the grain yield per plant decreased in OE lines but increased in C lines, with the grain yield per plant of C lines significantly surpassing that of OE lines (Fig. 4B, F).Furthermore, CRISPR/Cas9-mediated OsAAP7 knockout mutants exhibited enhanced NUtE compared to ZH11, while NUtE decreased in OE lines (Fig. 4G).

OsAAP7 regulated amino acids accumulation of phenylalanine, lysine and arginine in rice
To further explore the connection between OsAAP7 expression and amino acid composition in rice, we determined the concentrations of individual amino acids in the root, leaf blade and leaf sheath of transgenic lines during the vegetative stage, as well as in grain seed, leaf blade and leaf sheath during the reproductive stage.The results showed higher concentrations of Phe, Lys and Arg in OE lines compared to ZH11 (Fig. 5A-C).Conversely, concentrations of these amino acids were reduced in seedlings of C lines compared to ZH11 (Fig. 5A-C).Furthermore, during the reproductive stage, concentrations of Phe, Lys, and Arg in OE lines significantly exceeded with those in ZH11 (Fig. 5D-F), while the opposite trend was observed in C lines (Fig. 5D-F).Thus, our findings suggest a significant accumulation of amino acids Phe, Arg, and Lys in OsAAP7 OE lines, whereas concentrations decreased in C lines.
To further investigate the transport function of OsAAP7 protein for amino acids such as Phe, Lys, and Arg, transgenic seedlings were subjected to treatments with FITC-labeled Phe, Lys, or Arg for durations of 2 h, 6 h, or 10 h, respectively.The results, as illustrated in Supplementary Figure S9, demonstrated stronger fluorescence signals in OsAAP7 OE lines when treated with FITC-labeled amino acids compared to the control ZH11.Conversely, fluorescence signals were weaker in OsAAP7 C lines compared to ZH11 under the same treatments (Supplementary Fig. S9).
These findings strongly suggest that OsAAP7 protein plays a crucial role in the transport of amino acids, specifically Phe, Lys, and Arg in rice.The increased fluorescence in the OsAAP7 OE lines indicates higher uptake and transport of these amino acids, while the reduced fluorescence in the OsAAP7 C lines suggests a diminished capacity for amino acid transport compared to the control ZH11.This experimental evidence supports the notion that OsAAP7 is directly involved in the transport of Phe, Lys, and Arg in rice plants.

OsAAP7 mediated outgrowth of axillary buds under different concentrations of amino acids phenylalanine, lysine and arginine
To investigate OsAAP7 plays role in rice growth under different Phe, Lys and Arg concentrations, we cultured OsAAP7 transgenic lines and wild-type ZH11 under various concentrations of these amino acids for 35 d.We found that the plant height and fresh weight of OE lines significantly increased compared to ZH11 under 0.1 mM Arg treatment, while those of C lines remained relatively unchanged.However, OE lines showed a slight decrease in plant height and fresh weight under 0.3 mM Arg treatment, whereas C lines exhibited noticeable increases relative to ZH11 (Fig. 6A, Supplementary Fig. S10).Similar patterns were noted with Phe and Lys treatments (Fig. 6A, Supplementary Fig. S11, S12).
Furthermore, the length of the first bud in control ZH11 and OsAAP7 transgenic lines remained unchanged significantly under all concentrations of Arg.However, the second buds of OE lines exhibited accelerated growth compared to both control and C lines when treated with 0.1 mM Arg, yet significantly slower growth was observed compared to control and C lines under 0.2 mM and 0.3 mM Arg treatments (Fig. 6B, Supplementary Fig. S13).Similar trends were obtained under Lys and Phe treatments (Fig. 6B, Supplementary Fig. S14, S15).These findings suggest that the elongation of second axillary buds in OE lines was notably enhanced compared to ZH11 and C lines under low exogenous concentration of the amino acids Phe, Lys and Arg.Conversely, the axillary buds growth of OE lines was significantly suppressed compared to ZH11 under high concentrations of these amino acids treatments, while the axillary buds growth of C lines presented an opposite trend.

OsAAP7 regulated bud outgrowth by coordinating N, auxin and cytokinin pathways
To investigate the regulatory role of OsAAP7 in axillary bud outgrowth, RNA-seq analysis was conducted on control and OsAAP7 transgenic lines.A total of 312 differentially expressed genes (DEGs) were identified among ZH11, OE, and C lines (Fig. 7A).The reliability of the data was demonstrated among various samples of control and transgenic lines using principal component analysis (PCA) (Fig. 7B).Volcano plot analysis revealed 1086 upregulated and 621 down-regulated genes between ZH11 and OE lines, as well as 1983 up-regulated and 530 downregulated genes between ZH11 and C lines (Fig. 7C, D).GO and KEGG enrichment analyses of DEGs involved in the bud growth of OE and C lines of OsAAP7 were performed.The results indicated that DEGs in transgenic lines were predominantly enriched in secondary shoot formation, phenylpropanoid, lignin, and N compound processes through GO analysis (Fig. 7E) and in  beta-alanine metabolism, phenylpropanoid biosynthesis, amino acid degradation, cyanoamino acid metabolism, and phenylpropanoid biosynthesis through KEGG analysis (Fig. 7F).Additionally, GO enrichment revealed that DEGs in ZH11 and OE lines were mainly associated with amino sugar and aminoglycan processes (Supplementary Fig. S16A), while those in ZH11 and C lines were primarily related to aminoglycan, alpha-amino acid, and phenylpropanoid processes (Supplementary Fig. S16B).For the KEGG enrichment analysis, DEGs in ZH11 and OE lines exhibited enrichment mainly in phenylpropanoid biosynthesis, and beta-alanine, amino acid and N metabolisms (Supplementary Fig. S16C).Conversely, DEGs in ZH11 and C lines showed enrichment mainly in N, cyanoamino acid, starch, sucrose, and phenylalanine metabolisms (Supplementary Fig. S16D).These findings suggest that alterations in the expression of genes associated with pathways such as N metabolism, phenylpropanoid biosynthesis, and phytohormone regulation are crucial for modulating axillary bud outgrowth through changes in OsAAP7 expression levels (Supplementary Fig. S16).
The expression patterns of DEGs related to N and phytohormone pathways in buds were analyzed using heatmaps (Fig. 8).In the mutant lines of OsAAP7, there was a significantly higher expression of genes encoding ammonium transporters such as OsAMT1.1 and nitrate transporters like OsNPF3.1,OsNPF6.5, and OsNPF8.20 compared to the ZH11 wild type, while their expression was lower in the OE lines (Fig. 8A).Similarly, the expression of amino acid transporter genes like OsAAP14, OsANT1, OsANT3, and OsLHT1 was significantly higher in the mutant lines of OsAAP7 compared to ZH11, but lower than in the OE lines (Fig. 8B).Additionally, the signaling pathways of various plant hormones, including auxin (Fig. 8C, D), cytokinin (Fig. 8E), ethylene (Fig. 8F), gibberellin and strigolactone (Fig. 8G), jasmonic acid, abscisic acid, and brassinolide (Fig. 8H) were analyzed.In the auxin pathway, genes such as OsIAA17, OsIAA21, and OsIAA30 showed significant up-regulated in the OE lines compared to ZH11, while their expression was lower in the C lines (Fig. 8C, D).Moreover, in the cytokinin pathway, genes encoding cell division kinase activation enzymes like OsLOG and OsLOGL10 were significantly up-regulated in the C lines compared to ZH11, with the opposite trend observed in the OE lines (Fig. 8E).Furthermore, genes such as OsCKX4 [39] and other cell division kinase oxidase exhibited higher expression levels in the OE lines compared to ZH11, while their expression was lower in the C lines (Fig. 8E).The above results collectively suggested that the elongation of rice axillary buds in OsAAP7 altered expression lines is regulated by changes in the expression of genes involved in N and various phytohormone pathways.

Discussion
Knockout of OsAAP7 had the potential to increase yield by triggering tillering in rice Indica and japonica are two key subspecies in rice, differing significantly in their biology and yield [40].This study revealed 10 SNP sites variations in the promoter sequences of OsAAP7 across 521 wild rice varieties (Fig. 1A).Additionally, haplotypes (hap1 and hap2) clearly distinguished between indica and japonica (Fig. 1A), suggesting natural selection of the amino acid transporter OsAAP7 during rice evolution.Moreover, our research demonstrated that the expression level of OsAAP7 negatively regulates rice tillering and yield (Figs. 1 and 4).To date, only two AAP members, OsAAP3 and OsAAP5, have been reported to negatively regulate rice tillering [19,21].On the contrary, rice tillering and yield are positively regulated by other amino acid permeases, including OsAAP1, OsAAP4, OsAAP14 and OsAAP15 [18,20,23,24].This may be due to OsAAP7, OsAAP3 and OsAAP5 undergoing similar natural selection processes and performing analogous functions in rice.Crucially, knockout of OsAAP7 has the potential to increase yield by stimulating tillering in rice (Fig. 4), indicating that OsAAP7 offers a novel genetic resource for high-yield breeding application by CRISPR/Cas9 technology.

OsAAP7 protein transported phenylalanine, lysine and arginine in rice to inhibit outgrowth of axillary bud
In this study, we demonstrated the localization of OsAAP7 protein on the ER membrane (Supplementary Fig. S2), contrasting with plasma membrane localization of OsAAP1 and OsAAP5 [18,21].This variance indicates distinct membrane localization among rice amino acid transporters.Then, we observed heightened expression of OsAAP7 under treatment with basic and neutral amino acids through GUS staining and qRT-PCR (Supplementary Fig. S1).Our findings also revealed the transport of basic amino acids Lys and Arg, as well as neutral amino acid Phe, by OsAAP7 transgenic seedlings in FITClabeled amino acid uptake experiment (Supplementary Fig. S9).Moreover, yeast complementation experiment validated the ability of OsAAP7 protein to transport Lys and Phe (Fig. 3).Additionally, it showed that the concentrations of these amino acids Phe, Lys and Arg were significantly increased in the OsAAP7 OE lines compared to those in ZH11 by HPLC determination (Fig. 5), indicating that these amino acids can be facilitated by OsAAP7 transporter.Previous reports have implicated AAP members such as OsAAP3 and OsAAP5 in the transport of Arg and Lys [19,21].Furthermore, other AAP transporters like OsAAP1, OsAAP4 and OsAAP15 primarily mediate the transport of neutral amino acids Tyr, Val or Pro in rice [18,20,24].These results suggest Fig. 8 The expression pattern of DEGs participated in ammonium and nitrate transporter, amino acid transporter and phytohormone pathways in rice axillary buds of OsAAP7 transgenic lines.(A-H) Heatmaps display up-regulated and down-regulated genes.Red and blue indicate higher and lower expression of gene that the accumulation of basic amino acids particularly suppresses the outgrowth of axillary buds in rice, and OsAAP3, OsAAP5 and OsAAP7 may cooperatively controls axillary bud outgrowth primarily through the transport of Arg and Lys.Importantly, amino acid transporter OsAAP7 also facilitates the transport of the aromatic amino acid Phe (Fig. 3).

OsAAP7 negatively regulated axillary bud growth by coordinating N and phytohormone pathways
Tillering directly influences rice yield [41] and results from the elongation of axillary buds, which are influenced by environmental cues and internal factors [42,43].Our result indicated that overexpression of OsAAP7 inhibited the expression of transporters OsNPF6.5,OsNPF8.20,OsAAP14 and OsLHT1 in the N pathway (Fig. 8A, B).These transporters positively impact tillering in rice [23,[44][45][46], indicating that altered expression of OsAAP7 controls axillary bud outgrowth by affecting transporter expression in the N pathway.In the plant hormone pathway, auxin-related genes OsIAA17, OsIAA21 and OsIAA30 [47][48][49] are significantly upregulated in OE lines of OsAAP7 compared to ZH11, while the opposite pattern is showed in C lines (Fig. 8C, D).Besides, cell division kinase activator genes OsLOG and OsLOG10 are notably upregulated in C lines of OsAAP7 compared to ZH11 (Fig. 8E), suggesting that altered OsAAP7 expression regulates axillary bud outgrowth by influencing auxin and cytokinin pathways simultaneously.This differs slightly from amino acid transporter OsAAP5, whose expression level only affects the cytokinin pathway in regulating bud outgrowth [21].Based on these results, we propose that amino acid transporter OsAAP7 negatively affects tillering and yield by mediating the transport of basic and neutral amino acids, thereby influencing N, auxin and cytokinin pathways in rice.

Fig. 1
Fig. 1 OsAAP7 was divergent between indica and japonica.(A) SNPs of OsAAP7 promoter in 521 rice varieties.(B) Expression of OsAAP7 in the basal part of rice seedlings with three haplotypes.(C-E) Tiller number per plant, grain yield per plant and total weight with every variety of three haplotypes.(F) Expression of OsAAP7 in seedling axillary bud of ten individual varieties between Hap1 and Hap2.(G) Tiller number of these varieties between Hap1 and Hap2.Error bars represent the SD (n = 4).Various letters represent significant differences at P < 0.05.The rice varieties were planted in paddy at Guiyang under fertilizer of (N/P/K = 19%/7%/14%) 270 kg•ha -1

Fig. 2
Fig. 2 Staining and section analysis of OsAAP7 promoter-GUS transgenic lines.(A-E) OsAAP7 promoter-GUS staining was performed with root tip, lateral root, stem, leaf sheath and leaf blade of pOsAAP7-GUS transgenic plants during vegetative stage.Scale bars represent 1 mm.(F-J) OsAAP7 promoter-GUS staining was performed with root, stem, leaf sheath, leaf blade and young panicle of pOsAAP7-GUS transgenic plants during reproductive stage.Scale bars represent 1 mm.(K-L) Relative expression of OsAAP7 in various tissues of ZH11 was detected during vegetative and reproductive stages.(M-P) Paraffin sections of the stained root, leaf blade, leaf sheath and panicle.Scale bars represent 100 μm.Error bars represent the SD (n = 4)

Fig. 3 Fig. 4
Fig.3Yeast growth complementation assay with various amino acids.(A) YNB solid medium with 3 mM (NH 4 ) 2 SO 4 or amino acid as sole nitrogen source.The pDR196 (empty vector) was used with negative control.Photos were taken after 72 h of growth under 30 °C.(B) Growth rates were measured using yeast mutants with OsAAP7-pDR196 or empty vector pDR196 in (NH 4 ) 2 SO 4 , phenylalanine, lysine or leucine.Yeast were cultured in liquid media with 3 mM amino acids or 3 mM (NH 4 ) 2 SO 4 as sole N source for 50 h.OD600 was detected every 5 h.Error bars represent SD (n = 3)

Fig. 5 Fig. 6
Fig. 5 Influence of OsAAP7 on amino acid concentrations in various rice tissues of control, OE and C lines during vegetative and reproductive stages.(A-C) Different amino acid concentration of ZH11, OsAAP7-overexpressing lines (OsAAP7-OE) and OsAAP7 CRISPR lines (osaap7) in roots, leaf sheaths and leaf blades during vegetative stage.(D-F) Different amino acid concentration of ZH11, OsAAP7-overexpressing lines (OsAAP7-OE) and OsAAP7 CRISPR lines (osaap7) in grain seed, leaf sheath and leaf blade at reproductive stage.Error bars represent SD (n = 20, 20 seedlings were randomly selected from the three OE lines or three C lines respectively) for (A-F).*, ** and *** represent significant differences at P < 0.05, P < 0.01 and P < 0.001, respectively

Fig. 7
Fig. 7 RNA-seq analysis of the axillary buds of OsAAP7 transgenic lines.(A) Detection of differentially expressed genes (DEGs) in the axillary buds of OsAAP7-overexpressing lines (OE), OsAAP7 CRISPR lines (C) and wild-type ZH11 (adjusted P-value < 0.05 and fold change > 2).(B) Factor map of the PCA performed on 6 samples.Following PCA analysis of the 6 samples, three clusters were identified corresponding to OsAAP7 C lines (cluster 1, red), OsAAP7 OE lines (cluster 2, blue), and ZH11 (cluster 3, green).(C-D) Volcano plot of ZH11 compared to OsAAP7 OE and C lines.Orange colors represent upregulated genes and blue colors represent down-regulated genes.(E-F) Axillary buds of OsAAP7 transgenic lines by GO and KEGG analysis of intersections network between OsAAP7 OE lines and C lines